EP2268296A1 - Compositions et procédé permettant de diagnostiquer, de prévenir et de traiter la maladie d'alzheimer - Google Patents

Compositions et procédé permettant de diagnostiquer, de prévenir et de traiter la maladie d'alzheimer

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Publication number
EP2268296A1
EP2268296A1 EP08848333A EP08848333A EP2268296A1 EP 2268296 A1 EP2268296 A1 EP 2268296A1 EP 08848333 A EP08848333 A EP 08848333A EP 08848333 A EP08848333 A EP 08848333A EP 2268296 A1 EP2268296 A1 EP 2268296A1
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EP
European Patent Office
Prior art keywords
fcγriib
interaction
screening
inhibitor
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP08848333A
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German (de)
English (en)
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EP2268296B1 (fr
EP2268296A4 (fr
Inventor
Yong-Keun Jung
Sungmin Song
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SNU R&DB Foundation
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SNU R&DB Foundation
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Publication of EP2268296A4 publication Critical patent/EP2268296A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1716—Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to methods of diagnosing, preventing and treating Alzheimer' s disease based on the use of an inhibitor for the binding of amyloid- ⁇ to Fc ⁇ RIIb, and a method of screening the inhibitor. More particularly, the present invention relates to methods of diagnosing, preventing and treating Alzheimer' s disease using an inhibitor of the binding between amyloid- ⁇ and Fc ⁇ RIIb, which is selected from the group consisting of an Fc ⁇ RIIb protein or a variant thereof, an Fc ⁇ RIIb extracellular domain, an anti-Fc ⁇ RIIb antibody, a specific peptide and an Fc ⁇ RIIb-specific siRNA, and a method of screening the inhibitor.
  • an inhibitor of the binding between amyloid- ⁇ and Fc ⁇ RIIb which is selected from the group consisting of an Fc ⁇ RIIb protein or a variant thereof, an Fc ⁇ RIIb extracellular domain, an anti-Fc ⁇ RIIb antibody, a specific peptide and an Fc ⁇ RIIb-specific siRNA,
  • AD Alzheimer's disease
  • familial AD which has genetic links and runs in families
  • sporadic AD which develops in many people for no obvious reason.
  • AD patients typically have multiple cognitive deficiencies, which are manifested by memory impairment and psychological symptoms such as psychosomatic abnormalities, including increased anxiety and hypersensitivity.
  • Two pathological hallmarks are seen in the brains of patients who die of AD: senile plaques and neurofibrillary tangles.
  • Senile plaques are extracellular accumulations of proteins and dead cells, and are primarily composed of amyloid- ⁇ (A ⁇ ) peptides (Hardy, J. et al., Nat Neurosci. 1:355-358, 1998) .
  • a ⁇ amyloid- ⁇
  • a ⁇ is produced from amyloid precursor protein (APP) through proteolytic cleavage.
  • APP is cleaved by ⁇ - secretase (BACE) and ⁇ -secretase, yielding A ⁇
  • BACE ⁇ - secretase
  • a ⁇ A ⁇ is produced from amyloid precursor protein (APP) through proteolytic cleavage.
  • APP is cleaved by ⁇ - secretase (BACE) and ⁇ -secretase, yielding A ⁇ (Craven, R., Nat Rev. Neurosci. 2: 533, 2001; David, H. S. et al., Nat Rev. Neurosci. 2: 595-598, 2001; Yankner, B. A., Neuron 16: 921-932, 1996; Selkoe, D. J., Nature 399: A23-A31, 1999) .
  • BACE ⁇ - secretase
  • AD Alzheimer's disease
  • Current medications for AD include nicotinic receptor agonists, such as ABT-418; muscarinic receptor agonists, such as Xanomeline and YM-976; acetylcholine precursors, such as lecithin and acetyl-L- carnitine; metal chelators, such as desferrioxamine and clioquinol; beta-sheet breakers, such as iA ⁇ 5 and iA ⁇ ll; antioxidants, such as vitamin E, Ginkgo biloba, melatonin and idebenone; sAPP releasing agents, such as nicotine, acetylcholine and carbachol; ⁇ -secretase or ⁇ -secretase inhibitors, such as OM99-1, OM99-2, OM99-3 and Z-
  • Pro- apoptotic genes such as prostate apoptosis response-4 (Par-4), tau protein kinase 1 (GSK-3 ⁇ ) , Calsenilin/DREAM/KChIP3, and cell death-promoting gene 5 (DP5), are shown to be overexpressed or their activities are increased in neuronal cells cultured in the presence of
  • a ⁇ or neuronal cells from AD patients The blocking of the functions of the proteins reduces A ⁇ -induced neuronal death
  • Fc ⁇ RIIb Fc ⁇ receptor lib
  • Fc ⁇ RIIb receptor has recently been known to play a regulatory role in arthritis (Nakamura, A. et al., Biomed. Pharmacother. 58: 292-298, 2004) .
  • the involvement of Fc ⁇ RIIb in dementia and its potential as a therapeutic target for dementia have not been known.
  • Fc ⁇ RIIb serves as a receptor for A ⁇ as well as playing an immunoregulatory role.
  • Fc ⁇ RIIb acts as a protein mediating A ⁇ neurotoxicity and serves as a receptor in an A ⁇ -initiated toxic signaling pathway, through which Fc ⁇ RIIb binds A ⁇ as the first event of the toxic signaling in neuronal cells and transduces the cell death signal into the cells.
  • Fc ⁇ RIIb enhances A ⁇ deposition, associated with memory impairment in AD, within neuronal cells.
  • an Fc ⁇ RIIb protein or a variant thereof, an Fc ⁇ RIIb extracellular domain, an anti-Fc ⁇ RIIb antibody, an Fc ⁇ R ⁇ Ib-specific peptide and an Fc ⁇ RIIb-specific siRNA suppress neuronal cell death and prevent memory loss in subjects, thereby leading to the present invention.
  • the present invention provides a method of preventing and treating Alzheimer's disease, by inhibiting binding of A ⁇ to Fc ⁇ RIIb.
  • the present invention also provides an inhibitor for the binding of A ⁇ to Fc ⁇ RIIb.
  • the inhibitor includes an
  • Fc ⁇ RIIb protein or a variant thereof an Fc ⁇ RIIb extracellular domain, an anti-Fc ⁇ RIIb antibody, an Fc ⁇ RIIb- specific peptide, and an Fc ⁇ RIIb-specific siRNA.
  • the present invention further provides a method of screening an agent inhibiting the interaction between A ⁇ and Fc ⁇ RIIb.
  • the screening method includes screening an agent inhibiting the activity of Fc ⁇ RIIb, an agent suppressing the expression of Fc ⁇ RIIb, an agent inhibiting the transduction of the toxic signal of A ⁇ into neuronal cells through Fc ⁇ RIIb, and an agent inhibiting the interaction between A ⁇ and Fc ⁇ RIIb.
  • the ordinary skilled person may use computer software-based methods to identify the agent inhibiting the interaction between A ⁇ and Fc ⁇ RIIb.
  • the present invention still further provides a method of diagnosing Alzheimer' s disease comprising determining the expression level of Fc ⁇ RIIb.
  • the present invention still further provides a kit for diagnosing Alzheimer' s disease comprising reagents used for determining the expression level of Fc ⁇ RIIb.
  • the present invention still further provides a method of preventing and treating Alzheimer' s disease based on the use of the method of inhibiting the interaction between A ⁇ and Fc ⁇ RIIb.
  • a "peptide” means a molecule comprising two or more amino acids linked each other via peptide bond.
  • the peptide may be chemically synthesized or prepared by common genetic engineering technologies.
  • RNA or "small interfering RNA” means an RNA molecule binding to a particular tagert mRNA and knock-out the mRNA, and may be a double stranded RNA molecule having a homologous seguence selected from the target mRNA and consisting of 17 to 25 consecutive nucleotide or may be a short hairpin RNA (hereinafter referred as "shRNA”) comprising sequentially the homologous sequence, a loop and a complementary sequence of the homologous sequence thereby forming a stem-loop structure.
  • shRNA short hairpin RNA
  • the si RNA may be prepared by chemical synthesis of RNA oligonucleotides, in vitro transcription, cleavage of long dsRNA transcribed in vitro using RNase III or Dicer, expression through celluar transduction of plasmid or viral vector expressing siRNA or cellular transduction of PCR-derived siRNA expression cassette .
  • a “variant” or “mutant” means a peptide having conserved amino acids for keeping the function thereof and whose one or more non-essential amino acids are substituted with different amino acids but reserving the original function .
  • an “inhibitor of interaction” means a substance inhibiting the interaction between proteins and includes compositions comprising a protein or a peptide inhibiting interaction between proteins or an antibody against the protein or the peptide or expression inhibitors.
  • an “expression inhibitor” means a material inhibiting transcription or translation of a particular gene. It includes compositions comprising molecules commonly used for inhibition of expression of the gene such as siRNA, shRNA, microRNA (hereinafter referred as "miRNA”) and antisense oligonucleotide.
  • control group means an experimental group treated with only buffer solution used for solving a tested compound or a compound known to have no effect on a target.
  • Fc ⁇ RIIb chimeric protein means a chimeric (or fusion) protein of Fc ⁇ RIIb with an effector protein (a protein activating the expression, color development or color change of a specific protein or cellular signal transduction upon the interaction between Fc ⁇ RIIb and A ⁇ ) .
  • Fo ⁇ receptor lib extracellular domain means a portion of Fc ⁇ RIIb which is exposed to extracellular space.
  • Fo ⁇ receptor lib extracellular domain When recombinantly produced Fo ⁇ receptor lib extracellular domain is administrated to a subject, it can induce competitive inhibition with intrinsic Fey receptor libs.
  • an “antibody” means an immunoglobulin such as IgG, IgM and IgA or active fragment thereof such as Fab, F(ab')2 and ScFv, which binds to a target antigen specifically.
  • the antibody may be a polyclonal antibody prepared by immunizing a mammal such as a rabbit, a mouse, a rat or a goat through injecting a purified antigen along with an adjuvant; collecting blood from the mammal; and purifying antibodies using affinity column using protein A or G.
  • the antibody may be a monoclonal antibody prepared by obtaining spleen cells from the immunized mammal; producing hybridoma with myeloma cells; and culturing the hybridoma under a selection medium.
  • the monoclonal antibody may be produced as a humanized antibody prepared by analyzing a gene encoding said monoclonal antibody; determining complementary determining region (hereinafter referred as "CDR") ; reserving the CDR and replacing framework regions (hereinafter referred as "FRs”) with human FRs. More preferably, a human antibody may be used by screening human antibody cDNA library using phage display technologies, etc. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a method of preventing and treating AD by inhibiting the binding of A ⁇ to Fc ⁇ RIIb.
  • the present inventors identified a receptor for A ⁇ , which is the major pathological cause of AD, on neuronal cells, and, based on this finding, developed a method of preventing and treating AD by inhibiting the association between A ⁇ and Fc ⁇ RIIb.
  • the expression of Fc ⁇ RIIb was found to increase in neuronal cells exposed to A ⁇ (FIG. 1) .
  • a Fc ⁇ RIIb wild type and a Fc ⁇ RIIb variant were prepared and administered to neuronal cells along with A ⁇ .
  • cells treated with the Fc ⁇ RIIb variant were found to exhibit reduced cell death rates (FIG. 2) .
  • the present inventors prepared siRNAs to suppress the transcription of Fc ⁇ RIIb and RAGE, which is known to be a cell surface receptor for A ⁇ (FIG. 3a) .
  • the transcriptional suppression of RAGE expression did not result in any increase in cell survival, whereas all cells survived when the transcription of Fc ⁇ RIIb was suppressed (FIG. 3b) .
  • Fc ⁇ RIIb rather than RAGE is the direct cell surface protein for A ⁇ signaling.
  • Fc ⁇ RIIb extracellular domain was prepared, and neuronal cells exposed to A ⁇ were treated with this extracellular domain and examined for cell survival rates.
  • the increased expression of Fc ⁇ RIIb due to exposure to A ⁇ was suppressed (FIG. 4a), and the relative cell viability was increased to that of cells not exposed to A ⁇ (FIG. 4b) .
  • Fc ⁇ RIIb-mediated neuronal transduction of A ⁇ signaling was inhibited.
  • the in vivo distribution of A ⁇ and FcyRIIb was examined.
  • Oligomeric A ⁇ and Fc ⁇ RIIb were found to be co- localized in the brain tissue of Tg2576 mouse which is an animal model for AD, indicating that both of them are present in the same region (FIG. 5a) . Also, A ⁇ was expressed together with Fc ⁇ RIIb in brain specimens from AD patients, confirming that both of them are present in the same cells (FIG. 5b) . The transcriptional suppression of RAGE expression did not result in a decrease in the intracellular accumulation of A ⁇ , whereas the transcriptional suppression of Fc ⁇ RIIb expression markedly reduced intracellular A ⁇ accumulation (FIG. 6) . These results indicate that Fc ⁇ RIIb is involved in the intracellular accumulation of A ⁇ as well as in intracellular signal transduction of A ⁇ . Based on the above results, the binding between A ⁇ and Fc ⁇ RIIb was examined in vitro. The in vitro experiment revealed that A ⁇ binds to Fc ⁇ RIIb (FIGs. 7 and 8) .
  • a ⁇ bound to Fc ⁇ RIIb identified for the first time the structure of A ⁇ bound to Fc ⁇ RIIb using a computer program.
  • researchers have made many efforts to determine the structure of A ⁇ , but failed to crystallize A ⁇ .
  • a ⁇ is difficult to crystallize because it is present as amorphous aggregates, called amyloid plaques, in AD patients.
  • amyloid plaques amorphous aggregates
  • AD patients amorphous aggregates
  • soluble oligomers of A ⁇ play a major role in neuronal toxicity.
  • the three-dimensional structure of A ⁇ oligomers was determined by simulating the stable oligomerization of A ⁇ monomers using in silico methods (Urbane, B. et al., Proc. Natl.
  • the present inventors predicted the Fc ⁇ RIIb-bound structure of A ⁇ using the known three-dimensional structures of A ⁇ and Fc ⁇ RIIb. This predicted structure showed that the structures of A ⁇ and Fc ⁇ RIIb precisely fit together (FIG. 9) .
  • a ⁇ binds to an extracellular domain of Fc ⁇ RIIb.
  • a ⁇ is present in oligomeric forms, in which hydrophilic N- terminal regions are flexible and C-terminal regions aggregate to form an oligomer.
  • the present inventors found that extended N-terminal regions bind Fc ⁇ RIIb to exert cytotoxicity using Insight II/Affinity program (Acelrys Co. , USA) .
  • Trp87 and TrpllO of Fc ⁇ RIIIb interacts with a proline residue at 329 (Pro329) of IgG (Sondermann P. et al.,
  • the present inventors conducted an in silico simulation based on the above results. This simulation showed that a phenylalanine residue at position 4 of A ⁇ makes a strong hydrophobic interaction with two tryptophan residues, Trp92 and Trpll5, of Fc ⁇ RIIb, an aspartate residue at position 7 of A ⁇ makes a strong hydrophilic interaction with two lysine residues, Lysll ⁇ and Lysll ⁇ , of Fc ⁇ RIIb, a glutamine residue at position 3 of A ⁇ makes a relatively weak interaction with Tyrl65 of Fc ⁇ RIIb, and Arg5 and His6 residues of A ⁇ rarely interact with residues of Fc ⁇ RIIb.
  • the present inventors prepared peptides capable of inhibiting the binding between A ⁇ and Fc ⁇ RIIb.
  • the peptides were found to effectively inhibit the binding between A ⁇ and Fc ⁇ RIIb (FIGs. 10 and 11) .
  • mice The peptides were injected into the brain of mice, and the memory ability of mice was assessed. As a result, memory impairment, as seen in AD cases, was remarkably restored (FIG. 12) . Also, the mice treated with the peptide inhibitors displayed no accumulation of the full length A ⁇ i- 42 peptide in the brain (FIG. 13) .
  • the present invention provides interaction inhibitors inhibiting the interaction between A ⁇ and Fc ⁇ RIIb.
  • the interaction inhibitors may be selected from the group consisting of an Fc ⁇ RIIb protein or a variant thereof, an Fc ⁇ RIIb extracellular domain, an anti-Fc ⁇ RIIb antibody, a peptide inhibiting the binding between A ⁇ and Fc ⁇ RIIb, and an Fc ⁇ RIIb expression inhibitor including an Fc ⁇ RIIb-specific siRNA or an Fc ⁇ RIIb-specific antisense nucleotide.
  • An Fc ⁇ RIIb protein or a variant thereof competes with endogenous Fc ⁇ RIIb in neuronal cells for A ⁇ binding to inhibit the binding of A ⁇ to endogenous Fc ⁇ RIIb.
  • a cell death signal of A ⁇ is not transduced into neuronal cells, thus preventing cell death.
  • the Fc ⁇ RIIb protein has the nucleotide seguence of SEQ ID No. 35 and the amino acid sequence of SEQ ID No. 36, and variants thereof are also available.
  • an Fc ⁇ RIIb variant is prepared by replacing an isoleucine residue at 232 of human Fc ⁇ RIIb with threonine, and has the amino acid sequence of SEQ ID No. 37.
  • the variant is not specifically limited thereto, and any variant in which other residues of Fc ⁇ RIIb are mutated and which is able to modulate the signal transduction mediated by Fc ⁇ RIIb is available.
  • a ⁇ -induced neuronal cell death decreased ( FIG . 2 ) .
  • Fc ⁇ RIIb is composed of an extracellular domain and an intracellular domain, and the extracellular domain binds to A ⁇ .
  • the extracellular domain also competes with endogenous Fc ⁇ RIIb in neuronal cells for A ⁇ binding to thus inhibit the binding of A ⁇ to endogenous Fc ⁇ RIIb, thereby inhibiting neuronal cell death.
  • the Fc ⁇ RIIb extracellular domain may be any one derived from humans, mice, rats, or the like. Preferred is a human-derived extracellular domain of Fc ⁇ RIIb.
  • the Fc ⁇ RIIb extracellular domain may be produced using a method known to those skilled in the art, for example, through cloning into E.
  • the FcyRIIb extracellular domain is purified using a method described in Sondermann P. et al. [EMBO J., 18(5) : 1095-1103, 1999) .
  • the Fc ⁇ RIIb extracellular domain was found to increase the relative viability of neuronal cells exposed to A ⁇ to the same level as cells not exposed to A ⁇ (FIG. 4) .
  • An anti-Fc ⁇ RIIb antibody prepared using the entire region or extracellular domain of Fc ⁇ RIIb as an antigen, competes with A ⁇ for Fc ⁇ RIIb binding and thus inhibits the binding of A ⁇ to Fc ⁇ RIIb in neuronal cells.
  • the peptide is designed based on an amino acid sequence predicted as a binding site of A ⁇ to Fc ⁇ RIIb or vice versa (see FIG. 9), but is not limited thereto.
  • the peptide is a peptide or a mutant thereof, which consists of one to nine amino acids comprising phenylalanine at position 4 of SEQ ID No. 24, corresponding to the N- terminal region of A ⁇ .
  • the peptide is an amino acid, a peptide or a mutant thereof, which consists of one to nine amino acids, comprising tryptophan at position 5 of SEQ ID No.
  • peptides were designed to have sequences represented by SEQ ID Nos. 24 to 33 (peptides #1 to #10, respectively), but are not limited thereto.
  • SEQ ID Nos. 24 to 33 peptides #1 to #10, respectively
  • specific peptides #1, #4 and #9 effectively inhibited the binding between Fc ⁇ RIIb-CD40 and A ⁇ (FIG. 10) .
  • neuronal cells were treated with the peptides and A ⁇ , cells exhibited increased survival (FIG. 11) .
  • peptides were injected along with A ⁇ into the brains of mice, and mice were assessed for memory ability. Peptides #1 and #9 were found to restore A ⁇ -induced memory decline (FIG. 12) . The immunohistochemical analysis of the brain of experimental animals showed that peptide #1 completely inhibited intracellular accumulation of A ⁇ in neuronal cells (FIG. 13) .
  • Fc ⁇ RIIb expression inhibitor refers collectively to substances that specifically inhibit the transcriptional or translational expression of Fc ⁇ RIIb, and may include siRNAs, antisense nucleotides and compounds.
  • Fc ⁇ RIIb siRNAs are not limited to specific sequences, and any siRNA sequence capable of inhibiting the binding between A ⁇ and Fc ⁇ RIIb by suppressing the expression of Fc ⁇ RIIb may be used.
  • an Fc ⁇ RIIb-specific siRNA consists of sense and antisense sequences, which are represented by SEQ ID Nos . 11 and 12. Sense and antisense sequences are suitably annealed and inserted into a pSuper- neo vector (Oligoengine, USA) .
  • a siRNA expression vector useful in the present invention is not specifically limited, but is preferably prepared by introducing a nucleotide sequence corresponding to the siRNA into a commonly used siRNA expression vector, psiRNA (Invitrogen, USA), pRNA (GenScript, USA), psLentGene (USA), pSIREN (Clontech, USA), pU ⁇ shX (VectorCoreA, Korea), pSilencer (Ambion, USA) , or pSuper-neo (Loigoengine, USA) .
  • siRNA expression vector useful in the present invention is not specifically limited, but is preferably prepared by introducing a nucleotide sequence corresponding to the siRNA into a commonly used siRNA expression vector, psiRNA (Invitrogen, USA), pRNA (GenScript, USA), psLentGene (USA), pSIREN (Clontech, USA), pU ⁇ shX (VectorCoreA, Korea),
  • the vector may be introduced into the nucleus of cells in the form of pure plasmid DNA or a complex with a transfection reagent or a target delivery substance, or in the form of a recombinant virus vector.
  • Suitable viral vectors for use in the present invention include adenovirus, adeno- associated virus, and retrovirus including lentivirus.
  • Antisense nucleotides have been approved as drugs having potential for therapeutic application to various human diseases. According to the Watson-Crick base pairing rules, a nucleotide is annealed to (hybridized with) a complementary sequence of DNA, immature mRNA or mRNA to interrupt the transmission of genetic information. The specificity of antisense nucleotides to target sequences makes them exceptionally multi-functional. An antisense- nucleotide is a long chain of monomer units and thus can be readily synthesized to correspond to a target RNA sequence. Many reports have recently demonstrated the usefulness of antisense nucleotides as a biochemical tool in the study of target proteins (Rothenberg et al . , J. Natl.
  • an antisense oligonucleotide targeting cmyb has been used to completely eliminate myelogenous leukemia cells from the bone marrow of patients suffering from myelogenous leukemia (Gewirtz and Calabreta, U.S. Pat. No. 5,098,890).
  • Antisense nucleotides are known to have in vivo therapeutic efficacy on cytomegalovirus retinitis.
  • Antisense nucleotides to Fc ⁇ RIIb are not limited to specific sequences, but any antisense nucleotides inhibiting the binding between A ⁇ and Fc ⁇ RIIb by suppressing the expression of Fc ⁇ RIIb may be used.
  • the present invention provides a pharmaceutical composition for preventing or treating AD comprising the interaction inhibitor as an effective ingredient.
  • the present composition includes the effective ingredient in an amount of 0.0001 to 50 wt% based on the total weight of the composition.
  • the present composition may include one or more effective ingredients exhibiting functions that are the same as or similar to the interaction inhibitor.
  • the present composition may also include, in addition to the aforementioned effective ingredients, one or more pharmaceutically acceptable carriers for administration.
  • the pharmaceutically acceptable carrier may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and mixtures of two or more thereof.
  • the composition may further include other typical additives, such as antioxidants, buffers, and bacteriostatics.
  • diluents, dispersing agents, surfactants, binders and lubricants may be further added so as to be formulated into injectable formulations, such as solutions, suspensions and emulsions, pills, capsules, granules or tablets.
  • the carrier may be conjugated to a target site-specific antibody or other ligands so as to act specifically in the target site.
  • the composition may be desirably formulated according to each disease or ingredient using a proper method in the art or the method described in Remington's Pharmaceutical Science (updated version, Mack Publishing Company, Easton PA) .
  • the pharmaceutical composition of the present invention is administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or locally) .
  • the dosage may vary depending on the patient's weight, age, gender, health state and diet, administration time, administration mode, excretion rate, and severity of illness.
  • the daily dosage ranges from about 0.01 to 500 mg/kg, and preferably from 0.1 to 50 mg/kg.
  • the daily dosage may be taken as a single dose or divided into several doses.
  • the present invention provides a method of screening a substance inhibiting the binding between A ⁇ and Fc ⁇ RIIb, Fc ⁇ RIIb-mediated signal transduction, or the intracellular translocation of A ⁇ and F ⁇ RIIb. i) The present invention provides a method of screening an inhibitor of the interaction between A ⁇ and Fc ⁇ RIIb.
  • the screening method includes the steps of 1) adding a compound to be tested before, after or during the binding between all or part of Fc ⁇ RIIb and all or part of A ⁇ ; 2) measuring the binding degree between Fc ⁇ RIIb and A ⁇ ; and 3) determining whether the compound reduces the binding between A ⁇ and Fc ⁇ RIIb in comparison with a control.
  • the entire Fc ⁇ RIIb protein may have the sequence of SEQ ID No. 36, and the partial portion of Fc ⁇ RIIb may be an Fc ⁇ RIIb extracellular region which is represented by SEQ ID No. 38.
  • the entire A ⁇ protein may have the sequence of SEQ ID No. 34, and the partial portion of A ⁇ may be an N- terminal region of A ⁇ .
  • the screening may be carried out using various methods analyzing protein-protein interaction, which are known to those skilled in the art.
  • Such methods for analyzing the association between proteins include yeast two-hybrid system (Parida et al., Tuberculosis, 85: 347-355, 2005), immunoprecipitation (IP), BIAcoreTM, Fluorescence Energy Transfer (FRET), and GST-full down assay (Lee S. Y., Biochem. Biophys. Res. Commun., 334: 1445-1451, 2005), but the present invention is not limited thereto, and any known methods for analyzing the association between proteins may be used.
  • the present invention provides a method of screening an inhibitor of Fc ⁇ RIIb.
  • the screening method includes the steps of 1) contacting all or part of Fc ⁇ RIIb with a compound to be tested; 2) measuring the binding degree of the compound to Fc ⁇ RIIb; and 3) determining whether the compound has high binding affinity to Fc ⁇ RIIb in comparison with a control.
  • the entire Fc ⁇ RIIb protein may have the sequence of SEQ ID No. 36, and the partial portion of Fc ⁇ RIIb may be an Fc ⁇ RIIb extracellular region, which is represented by SEQ ID No. 38.
  • the screening may be carried out using various methods analyzing protein-compound interaction, which are known to those skilled in the art. Such methods include MALDI-TOF, but the present invention is not limited thereto, and any known methods for analyzing the association between a protein and a compound may be used.
  • the present invention provides a method of screening a substance inhibiting the expression of Fc ⁇ RIIb.
  • the screening method includes the steps of 1) treating a brain cell culture with a compound to be tested; 2) measuring the expression level of Fc ⁇ RIIb in the brain cell culture; and 3) determining whether the compound inhibits Fc ⁇ RIIb expression in comparison with a control.
  • B103 cells or primary neuronal cells from the cerebral cortex may be used, but the present invention is not limited thereto, and any known cell lines expressing Fc ⁇ RIIb may be used.
  • the Fc ⁇ RIIb expression may be assessed using RT-PCR, an immunoassay, and the like, but the present invention is not limited thereto, and any known methods for measuring the amount of a transcript or a protein translated therefrom may be used.
  • the present invention provides a method of screening a substance inhibiting the intracellular translocation of A ⁇ and Fc ⁇ RIIb.
  • the screening method includes the steps of 1) treating a brain cell culture with A ⁇ and a compound to be tested; 2) detecting the intracellular level of A ⁇ in the brain cell culture; and 3) determining whether the compound inhibits the intracellular translocation of A ⁇ in comparison with a control.
  • B103 cells or primary neuronal cells from the cerebral cortex may be used, but the present invention is not limited thereto and any known cell lines expressing Fc ⁇ RIIb may be used.
  • the intracellular translocation of A ⁇ may be assessed using antibodies, compounds and peptides binding specifically to A ⁇ or FcyRIIb.
  • a ⁇ and FcyRIIb may be detected using a protein conjugated to a fluorescent, colorimetric or radioactive protein, compound or peptide.
  • a ⁇ and Fc ⁇ RIIb may be detected using fluorescence detection, radioactive detection and colorimetric detection apparatuses .
  • the present invention provides a method of screening a substance inhibiting the interaction between A ⁇ and Fc ⁇ RIIb using an Fc ⁇ RIIb chimeric protein.
  • the screening method includes the steps of 1) treating a cell line expressing an Fc ⁇ RIIb chimeric protein with A ⁇ and a compound to be tested; 2) measuring the activity of the Fc ⁇ RIIb chimeric protein; and 3) determining whether the compound inhibits the activity of the chimeric protein in comparison with a control.
  • the Fc ⁇ RIIb chimeric protein is a receptor, and any protein capable of measuring the force of interaction between A ⁇ and Fc ⁇ RIIb, as determined through the expression, color development or color change thereof, or the cellular signal transduction mediated thereby, may be fused to Fc ⁇ RIIb.
  • the Fc ⁇ RIIb chimeric protein may be created by linking Fc ⁇ RIIb to a specific protein, of which the expression, color development, color change or cellular signal transduction is stimulated upon the interaction between Fc ⁇ RIIb and A ⁇ .
  • CD-40 which activates cellular signal transduction, was linked to Fc ⁇ RIIb.
  • the transmembrane protein CD-40 mediating intracellular signal transduction was used, but a protein such as tyrosine receptor kinase (TRK) , which contains both a transmembrane domain and a cytoplasmic domain, is preferred.
  • TRK tyrosine receptor kinase
  • the present invention is not limited thereto .
  • the present invention provides a method of screening a substance inhibiting the interaction between A ⁇ and F ⁇ RIIb using a software program.
  • the screening method includes the steps of 1) inputting information about the structure of a compound to be tested into a software program; and 2) determining whether the compound inhibits the binding between A ⁇ and Fc ⁇ RIIb using the software program.
  • the software program useful in the method may be selected from the group consisting of DOCKTM, FlexXTM, and AffinityTM.
  • the present inventors employed an Affinity Program (InsightII, Accelrys Inc) .
  • a compound inhibiting A ⁇ -Fc ⁇ RIIb interaction may be determined based on 1) the protein structure of Fc ⁇ RIIb, containing amino acids corresponding to glutamic acid at position 64, tryptophan at 132, tryptophan at 155, lysine at 156 and lysine at 158 of SEQ ID No. 36, and 2) the protein structure of A ⁇ , containing amino acids corresponding to glutamic acid at position 3, phenylalanine at 4, histidine at 6 and aspartic acid at 7 of SEQ ID No. 34.
  • the present invention provides a method for screening of an inhibitor of intracellular kinase acting downstream of Fc ⁇ RIIb.
  • the present inventors confirmed that apoptosis occurred via activations of various intracellular kinases when oligomeric A ⁇ bound to Fc ⁇ RIIb through a series of experiments using expression inhibitors and activity inhibitor of the kinases.
  • the preferred intracellular kinase is Syk, Btk, Lyn, IP3K, JNK, GSK or IMPase, but not limited thereto.
  • the screening of an inhibitor inhibiting activity of the intracellular kinases may be performed by methods well- known in the art. Particularly, in a preferred embodiment the method is selected from a group consisting of: i) Reacting an intracellular kinase, a substrate specific for the kinase and test compounds, and selecting a test compound reducing the extent of phosphorylation of said kinase comparing to control which is not treated with the test compound; ii) Reacting an intracellular kinase and test compounds, and selecting a test compound which is binding to the kinase; and iii) Reacting an intracellular kinase, a substrate specific for the kinase and test compounds, and selecting a test compound inhibiting the interaction between the kinase and the substrate comparing to control which is not treated with the test compound.
  • the extent of phosphorylation of the kinase may be determined by various methods, for example, isotopes such as 32 P or 33 P, an agarose electrophoresis analysis in which the charge of a phosphorylated substrate changes by -2 or detection using antibodies specific for phosphorylated kinases may be used.
  • the present invention provides a method of diagnosing Alzheimer' s disease by measuring the expression level of Fc ⁇ RIIb.
  • the expression level of Fc ⁇ RIIb may be detected using any known methods capable of measuring the expression level of the Fc ⁇ RIIb protein. Examples of such methods include, but are not limited to, an immunoassay with an antibody binding specifically to Fc ⁇ RIIb, and RT-PCR and Northern blotting with nucleic acid molecules capable of complementarily binding to the FcyRIIb gene.
  • the present invention provides a kit for diagnosing Alzheimer' s disease by measuring the expression level of Fc ⁇ RIIb.
  • the diagnostic kit may include DNA, RNA and a protein, binding specifically to Fc ⁇ RIIb, a buffer, a standard antibody, a secondary antibody labeled with an enzyme catalyzing a colorimetric reaction or a fluorescent substance, and a substance for color development. Also, when a compound binding specifically to Fc ⁇ RIIb is used, this compound is used in a form in which it is conjugated to a fluorescent or colorimetric label, which may be visually detected.
  • the present invention also provides a method of diagnosing Alzheimer's disease using the diagnostic kit. The method includes the steps of 1) collecting a specimen from a subject; 2) reacting the specimen with a substance binding specifically to Fc ⁇ RIIb and washing the specimen; and 3) measuring the amount of the specifically bound substance.
  • step 3 when an antibody specific to Fc ⁇ RIIb is used, the antibody is allowed to react with a secondary antibody conjugated to a fluorescent substance, is washed, and is analyzed using a fluorescence microscope or scanner.
  • the bound compound may be quantified in a bound or separated state.
  • the present invention provides a therapeutic agent for treating AD comprising an activity inhibitor or an expression inhibitor of intracellular kinase acting downstream of Fc ⁇ RIIb.
  • the intracellular kinase is preferably Syk, Btk, Lyn, IP3K, JNK, GSK or IMPase, but not limited thereto.
  • the activity inhibitor may be one known to the art, and a compound screened in the above method of vii, IV may be used.
  • LY294002 As the activity inhibitor, LY294002, SP600125, LiCl, L-690,330 or piceatannol, etc. may be used, but not limited thereto.
  • antisense oligonucleotides, siRNA, miRNA or shRNA specific for the intracellular kinases may be used, but not limited thereto.
  • the therapeutic agent of the present invention is administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or locally) .
  • the dosage may vary depending on the patient's weight, age, gender, health state and diet, administration time, administration mode, excretion rate, and severity of illness.
  • the daily dosage ranges from about 0.01 to 500 mg/kg, and preferably from 0.1 to 50 mg/kg.
  • the daily dosage may be taken as a single dose or divided into several doses.
  • FIG. 1 shows the Fc ⁇ RIIb expression, increased upon exposure to A ⁇ i- 42 , which was detected using RT-PCR (a) , Western blotting (b) and immunostaining (c) : A ⁇ : A ⁇ i_ 42 ; Bapta: Bapta-AM; CaIp.: Calpeptin; Asc. : Ascorbic acid; and
  • Tuni tunicamycin.
  • FIG. 2 shows neuronal cell death induced by overexpression of Fc ⁇ RIIb and an Fc ⁇ RIIb mutant.
  • FIG. 3a is a photograph showing results of western blot analyzing effect of siRNA against Fc ⁇ RIIb and a siRNA against RAGE
  • FIG. 3b is a graph comparing degree of cell death of various cell lines: pSuper-Neo: mock vector; siFc ⁇ RIIb #1: siRNA-expressing cell line #1; siFc ⁇ RIIb #2: siRNA-expressing cell line #2; siRAGE #1: siRNA-expressing cell line #1; and siRAGE #2: siRNA-expressing cell line #2) .
  • FIG. 4a is a photograph showing western blotting analyzing effect of treatment of Fc ⁇ RIIb extracellular domain (ED), and FIG. 4b is a graph comparing a relative ratio of cell death treated with A ⁇ and/or Fc ⁇ RIIb ED:
  • FIG. 5a and 5b are photographs showing the results of immunohistochemical analysis for Fc ⁇ RIIb expression levels in the brains of Tg2576 mice, and AD patients, respectively.
  • FIG. 6 is a photograph showing the results of immunostaining for intracellular accumulation of A ⁇ i-42 in siFc ⁇ RIIb-transfected cells.
  • FIG. 7a is a photograph showing the results of an in vitro binding assay between Fc ⁇ RIIb and A ⁇ i_ 4 2 using gel shift assay
  • FIG. 7b is a graph showing whether Fc ⁇ RIIb binds to A ⁇ i_ 4 o and A ⁇ i- 42 using surface plasraon resonance analysis :
  • FIG. 8a is a schematic diagram an Fc ⁇ RIIb-CD40 chimeric protein
  • FIG. 8b is a graph shwong the stimulation of NF- ⁇ B activation when A ⁇ i_ 42 binds to Fc ⁇ RIIb.
  • FIG. 9 is a computer simulation showing the predicted binding site structure of A ⁇ i_ 42 and Fc ⁇ RIIb.
  • FIG. 10a depicts the sequences of peptides antagonizing the binding between A ⁇ i- 42 and Fc ⁇ RIIb and FIG. 10b is a graph showing degree of inhibition among the antagonistic peptides.
  • FIG. 11a and lib are graphs showing the effects of the peptides inhibiting the binding between A ⁇ i_ 42 , Fc ⁇ RIIb on A ⁇ -induced neurotoxicity in hippocampal neurons and cortical neurons, respectively.
  • FIG. 12 is a series of graphs showing the inhibitory effects of the peptides inhibiting the binding between A ⁇ i_ 42 and Fc ⁇ RIIb on memory decline: a and b: Y-maze test; and c and d: passive avoidance test.
  • FIG. 13 is a series of photographs showing the results of immunostaining for intraneuronal accumulation of A ⁇ i- 42 in neurons treated with peptides inhibiting the binding between A ⁇ i_ 42 and Fc ⁇ RIIb.
  • FIG. 14a is a graph showing the results of experiments for investigating whether siRNAs against various intracellular kinases can inhibit apoptosis of neuronal cells, which is induced by overexpression of Fc ⁇ RIIb, and
  • FIG. 14b is a graph showing the result of experiments for investigating whether the siRNAs can inhibit apoptosis of neuronal cells, which is induced by interaction between A ⁇ _
  • FIG. 15a is a graph showing the results of experiments for investigating whether inhibitors of various intracellular kinase can inhibit apoptosis of neuronal cells, which is induced by overexpression of Fc ⁇ RIIb
  • FIG. 15b is a graph showing the result of experiments for investigating whether the inhibitors can inhibit apoptosis of neuronal cells, which is induced by interaction between A ⁇ i- 42 and Fc ⁇ RIIb.
  • EXAMPLE 1 Gene expression profiling using DNA microarray
  • Neuronal cells were isolated from the cerebral cortex of 16 day-old rat embryos and cultured. The primary- cultured cortical neuronal cells were exposed to 5 ⁇ M of A ⁇ (500 ⁇ M in PBS; Sigma, USA) for 24 hrs . Total RNA was isolated from the cells using TRIZOL Reagent (GIBCO-BRL, USA) according to the manufacture's protocol. Gene expression was analyzed using DNA microarray filters (GF300, GF301, GF302, Invitrogen, USA) containing 17,000 rat cDNAs according to the manufacturer's instruction. Results obtained from three independent experiments were statistically analyzed using the Pathway3TM software program (ResgenTM, Invitrogen, USA) .
  • RNA for reverse transcription was isolated using TRIZOLR Reagent (Invitrogen, USA) .
  • cDNAs were synthesized through reverse transcription, which was carried out using 5 ⁇ g of total RNA and ImProm-IITM Reverse Transcriptase (Promega, USA) according to the manufacturer's protocol.
  • RT-PCR was performed using the following primers: Fc ⁇ RIIb-5 ' -EcoRI primer (5'- CGCGGAATTCGATGGACAGCAACAGGACT-3' : SEQ ID No.
  • Fc ⁇ RIIb-3'- Kpnl primer (5'-CGGGTACCATAATGTGGTTCTGGTAGTC-S' : SEQ ID NO. 2), Fc ⁇ RI-RT-5' primer (5'-TTGGTGAACACAGTTCTCTATGTGAAAATAC- ACAGGCTGC-3' : SEQ ID No. 3), Fc ⁇ RI-RT-3' primer (5'- CTATCTTACAGTGGCTGTTACTTCTTCATACACGTCATCGCT-S': SEQ ID NO. A), Fc ⁇ RIIa-RT-5' primer (5'-GCCGATTTCTGCCTAGTGATGTGCCTCCTGTTTGCA- GTGG-3': SEQ ID No.
  • ⁇ -actin was used as an internal control, and was amplified with the ⁇ -actin sense primer (5'-GCGTCCACCCGCGAG-S' : SEQ ID NO. 7) and the ⁇ -actin anti-sense primer (5'-TATAGCAGGGTCAAC-S' : SEQ ID No. 8) .
  • PCR was carried out in a total volume of 50 ⁇ 1 using one-fifth of the reverse transcription reaction solution as a template.
  • PCR conditions included denaturation at 95 ° C for 5 min, and 10, 15, 20 or 25 cycles of denaturation at 95 ° C for 60 sec, annealing at 56 ° C for 60 sec and extension at 72 ° C for 60 sec, followed by final extension at 72 ° C for 7 min.
  • EXAMPLE 2-2 Western blot analysis
  • Rat B103 neuronal cells were treated with a calcium chelator (BAPTA-AM, 5 ⁇ M; EGTA, 1 mM) , a calpain protease inhibitor (calpeptin, 10 ⁇ M) , and an antioxidant (ascorbic acid, 5 ⁇ M) for 2 hrs, and exposed to A ⁇ for 48 hrs. Then, cells were lysed with a sampling buffer (10% glycerol, 2% SDS, 62.5 mM Tris-HCl, 2% ⁇ -mercaptoethanol, pH 6.8).
  • BAPTA-AM calcium chelator
  • calpeptin 10 ⁇ M
  • an antioxidant ascorbic acid, 5 ⁇ M
  • the cell lysates were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred onto a nitrocellulose membrane.
  • Western blotting was performed with the primary antibody, monoclonal K9.631 (a gift from Dr. Hammerling, Memorial Sloa Kettering Cancer Center, NY) and goat anti-mouse IgG antibody-conjugated horseradish peroxidase as a secondary antibody (Santa Cruz Biotechnology, USA) .
  • a control was incubated with anti- ⁇ -tubulin antibody (T5168) (Sigma, USA) and the same HRP-conjugated secondary antibody.
  • the Western blotting showed that the A ⁇ -induced Fc ⁇ RIIb expression was not suppressed upon treatment with the calcium chelators, calpain protease inhibitor and antioxidant (FIG. Ib) .
  • the A ⁇ -induced Fc ⁇ RIIb expression seems to occur in a specific manner.
  • the Fc ⁇ RIIb expression increased even upon the blocking of the action of calcium and active oxygen species, which mediate the toxic signaling of A ⁇ , indicating that A ⁇ acts upstream of A ⁇ signaling to induce Fc ⁇ RIIb expression.
  • Example 2-1 B103 cells were exposed to PBS, tunicamycin (Tuni), which inhibits N-glycosylation as post-translational modification of proteins, and A ⁇ for 48 hrs. Cells were fixed, probed with the primary antibody monoclonal K9.631 (Memorial Sloa Kettering Cancer Center, NY) , and observed under a fluorescence microscope (Leica DMRBE, Germany) . This test was carried out as described in Song et al., Molecular cell, 12(3), 553-563, 2003. When B103 cells were exposed to A ⁇ , the expression of Fc ⁇ RIIb increased (FIG. Ic) . These results were consistent with those of the DNA microarray analysis in Example 1.
  • An FcyRIIb expression vector and an Fc ⁇ RIIb mutant expression vector were constructed and transfected into rat neuronal B103 cells.
  • the expression vectors were prepared as follows.
  • the rat FcyRIIb gene was amplified by performing PCR using a rat brain cDNA library (Invitrogen,
  • Fc ⁇ RIIb-3'-.FfpnI primer (5 ' -CGGGTACCATAATGTGGTTCTGGTAGTC-S ' : SEQ ID No. 2) .
  • PCR was carried out in a total volume of 100 ⁇ 1 using 20 pmol of each primer. PCR conditions included denaturation at 95 ° C for 5 min, and 30 cycles of denaturation at 95 ° C for 60 sec, annealing at 56 ° C for 60 sec and extension at 72 ° C for 60 sec, followed by final extension at 72 ° C for 7 min.
  • the amplified rat Fc ⁇ RIIb gene was inserted into pEGFP-Nl (Clonetech, USA) , and the resulting vector was designated "pFc ⁇ RIIb".
  • Fc ⁇ RIIb (I232T) mutant was prepared through PCR using Fc ⁇ RIIb [I232T]-5' primer ( ⁇ '-GCTGTCGCTGGAACTGTAGCTGCC-S': SEQ ID No. 9) and Fc ⁇ RIIb [I232TJ-3' primer (5'-GGCAGCTACA- GCAGTTCCAGCGACAGC-3' : SEQ ID No. 10) .
  • PCR was carried out in a total volume of 50 ⁇ 1 using 10 pmol of each primer. PCR conditions included denaturation at 95 ° C for 5 min, and 30 cycles of denaturation at 95 ° C for 5 min, annealing at 5 ⁇ ° C for 60 sec and extension at 72 ° C for 10 sec, followed by final extension at 72 ° C for 30 min.
  • the amplified rat Fc ⁇ RIIb [I232T] mutant gene was inserted into pEGFP-Nl (Clonetech, USA) , and the resulting vector was designated "pFc ⁇ RIIb [I232T] ".
  • This vector was digested with Dpnl , and the excised mutant gene was subjected to DNA sequencing, which was performed by the COSMO Company (Korea) . Then, B103 cells were transfected with 300 ng of pEGFP, 900 ng of pcDNA3 (void vector) , 900 ng of pFc ⁇ RIIb, and 900 ng of pFc ⁇ RIIb [I232T] using lipofectamine (Invitrogen, USA) according to the manufacturer's instructions. Cells were then exposed to 5 ⁇ M of A ⁇ and phosphate buffered saline (PBS) for 48 hrs. Cell viability was estimated under a fluorescence microscope based on the morphology of green fluorescent protein (GFP) -positive cells (expressing GFP through pEGFP introduction) .
  • GFP green fluorescent protein
  • Fc ⁇ RIIb-overexpressing B103 cells exhibited increased cell death, whereas neuronal cell death was inhibited in B103 cells transfected with the Fc ⁇ RIIb mutant expression vector (FIG. 2) .
  • Fc ⁇ RIIb mutant expression vector FIG. 2
  • siRNAs Small interfering RNAs (siRNAs) inhibiting the expression of Fc ⁇ llb and receptors for advanced glycation end-product (RAGE) , which is known as a cell surface receptor of A ⁇ , were constructed, and their effects on cell death were compared to each other.
  • a siRNA duplex was formed by hybridizing sense and antisense complementary RNA oligonucleotides, listed in the following Table 1, and was inserted into pSuper-neo (Oligoengine, USA) .
  • the siRNA expression vectors thus constructed were individually transfected into B103 cells using lipofectamine (Invitrogen, USA) according to the manufacturer's protocol.
  • transfected cells were designated "pSuper- neo”, “psiFc ⁇ RIIb#l”, “psiFc ⁇ RIIb#2”, ⁇ psiRAGE#l", and "psiRAGE#2". Then, transfected cells were subjected to Western blot analysis.
  • Western blotting was performed as described in Example 2-2 with anti-Fc ⁇ RIIb antibody (primary antibody: K9.361; secondary antibody: goat anti- mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA) ) , anti-RAGE antibody (primary antibody: Sc8230 (Santa Cruz Biotechnology, USA) ; secondary antibody: donkey anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA)), and anti- ⁇ -tubulin antibody (primary antibody: T5168 (Sigma, USA) ; secondary antibody: goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA) ) .
  • anti-Fc ⁇ RIIb antibody primary antibody: K9.361
  • secondary antibody goat anti- mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA)
  • anti-RAGE antibody primary antibody: Sc8230 (Sant
  • siRNAs were found to completely suppress the expression of Fc ⁇ RIIb and RAGE (FIG. 3a) .
  • the transformed B103 cells were exposed to 5 ⁇ M of A ⁇ or PBS for 48 hrs, and their viability was evaluated. Cell survival was assessed through trypan blue exclusion, Hoechst staining (Sigma, USA) , and Annexin V labeling (Promega, USA) . The survival of effector cells and cells introduced with pEGFP
  • EXAMPLE 5 The effect of Fc ⁇ RIIb extracellular domain (ED) on neuronal cell death
  • Fc ⁇ RIIb extracellular domain was purified as described in Sondermann et al . , EMBO J., 18:1095-1103, 1999.
  • Neuronal B103 cells or primary-cultured neuronal cells were exposed to 5 ⁇ M of A ⁇ for 48 hrs . Then, cells were treated or not treated with 100 ⁇ g of the purified Fc ⁇ RIIb ED, or treated or not treated with 100 ⁇ g of bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • B103 cells were subjected to Western blotting, which was performed with anti-Fc ⁇ RIIb antibody as described in Example 2-2.
  • the primary neuronal culture was evaluated for cell survival as described in Example 3.
  • the Fc ⁇ RIIb ED Compared to BSA treatment, the Fc ⁇ RIIb ED was found to completely inhibit A ⁇ signaling in A ⁇ -exposed cells (FIG. 4), indicating that the Fc ⁇ RIIb ED is an extracellular receptor of A ⁇ . Thus, the Fc ⁇ RIIb ED may have potential as a target for inhibiting the neurotoxic signaling initiated by A ⁇ .
  • EXAMPLE 6 Immunohistochemical assay The transgenic mouse used in this test was Tg2576 (18 to 24 months old, female) , which contained the human APP695 with the double mutation Lys670 ⁇ Asn and Met671 ⁇ Leu (K670N, M671L) , which was found in a large Swedish family suffering from the early onset of Alzheimer's disease (Hsiao et al . , Science, 274:99-102, 1996) .
  • the mouse was anesthetized with 7% chloral hydrate, and perfused transcardially with 4% phosphate-buffered paraformaldehyde (PFA; Sigma, USA) .
  • PFA phosphate-buffered paraformaldehyde
  • the brain was excised and immersed in PFA for 48 hrs . Then, the brain was cut into serial coronal sections on a freezing microtome. The sections were mounted on glass slides, dried, and fixed again with 4% PFA for 15 min. The sections were incubated in methanol, containing 3% H2O2 for 5 min, to remove endogenous peroxidase activity. Then, the brain sections were washed, immersed in 0.5% Triton X-IOO for 30 min before being reacted with the primary antibody, and incubated with 1% bovine serum albumin for 1 hr.
  • Specimens from fifteen patients neuropathologically diagnosed as having AD were donated from McLean Hospital (Harvard Brain Tissue Resource Center, Belmont, Massachusetts) and Ohio state university (Columbus, Ohio) . All tissues were confirmed through clinical records and neuropathological examinations .
  • AD patient samples were stained with anti-A ⁇ monoclonal antibody (4G8:Signet, USA), anti-PHF-1 antibody (gift from Dr. Davis, Albert Einstein College of Medicine, NY) .
  • EXAMPLE 7 Evaluation of intracellular A ⁇ accumulation in B103 cells transfected with siRNA expression vectors psiFc ⁇ RIIb #1 cells or psiRAGE #1 cells, prepared in Example 4, were exposed to 100 nM of A ⁇ or PBS for 12 hrs, and immunostained with anti-A ⁇ antibody (4G8; Signet, USA) according to the same method as in Example 2-3. Intracellular A ⁇ accumulation was strongly inhibited in psiFc ⁇ RIIb cells but was maintained in psiRAGE cells, indicating that Fc ⁇ RIIb mediates the intracellular accumulation of A ⁇ in neurons (FIG. 6) . Thus, psiFc ⁇ RIIb of Fc ⁇ RIIb mutants may be useful in inhibiting intraneuronal A ⁇ accumulation.
  • EXAMPLE 8 Jn vitro assay for the binding between Fc ⁇ RII and ⁇ i_ 42
  • EXAMPLE 8-1 Gel shift assay 5 ⁇ M of A ⁇ was mixed with 20 ⁇ g of Fc ⁇ RIIb-ED or 20 ⁇ g of BSA in vitro, and was incubated at 37 ° C for 3 hrs. The reaction mixture was incubated with anti-Fc ⁇ RIIb polyclonal antibody or anti-GST antibody for 2 hrs, and then with Protein G for 3 hrs (binding solution: 50 mM Tris-HCl, pH 7.4, 1 mM DTT, 0.5 mM EDTA, 0.01% Triton X-100, 0.5 mg/ml bovine serum albumin, 10% (v/v) glycerol, protease inhibitors cocktail, several concentrations of NP-40) .
  • the beads were washed three times and subjected to Western blotting to assess the association between Fc ⁇ RII and A ⁇ i- 42 - Western blotting was carried out with K9.361 antibody and anti-A ⁇ antibody (primary antibody: 71-5800 (Zymed, USA) ; secondary antibody: goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA) .
  • a ⁇ was found to directly bind to Fc ⁇ RIIb-ED (FIG. 7a) .
  • the present inventors performed surface plasmon resonance assay.
  • the inventors attached BSA and Fc ⁇ RIIb- ED into CM5 chip (GE Healthcare, USA) and then determined binding activity with A ⁇ i_ 42 using Biacore 3000 (GE Healthcare, USA) (FIG. 7b) .
  • a ⁇ i_ 40 showed weaker affinity than A ⁇ i- 42 . This suggests that Fc ⁇ RIIb binds to A ⁇ i- 42 preferentially.
  • EXAMPLE 9 Evaluation of the binding between Fc ⁇ RIIb-CD40 chimera and A ⁇
  • Fc ⁇ RIIb binds to A ⁇ in a specific manner, an extracellular domain of Fc ⁇ RIIb was genetically fused to CD40, consisting of a transmembrane domain and a cytoplasmic domain.
  • the resulting Fc ⁇ RIIb- CD40 fusion gene was expressed in NIH3T3 cells to increase NF-KB activity, a signal transducer of CD40, when the fusion protein binds to A ⁇ .
  • the chimeric gene was constructed as follows: A rat Fc ⁇ RIIb extracellular region and human CD40 transmembrane and cytoplasmic domains were amplified by performing PCR with Fc ⁇ RIIb-ED-5 ' -Nhel primer (5 ' -GCTAGCGCTATGGACAGCAACAGGACT-S ': SEQ ID No. 15), Fc ⁇ RIIb-ED-3'-Hi ⁇ d III primer (S'-AAGCTTGGGAGGCAACGAACTGC- TGGATTT3': SEQ ID No. 16), CD40-TM+cyto-5 ' primer (5 1 - CCCAAGCTTGGGGCCCTGGTGGTGATCCCCATC-3' : SEQ ID No.
  • a computational study revealed the assembly of A ⁇ monomers into a globular soluble oligomeric structure, in which N- terminal tails are exposed to the exterior and C-terminal hydrophobic regions aggregate to form an oligomer (Urbane, B. et al. Proc.Natl. Acad. Sci. U. S. A. 101:17345-17350, 2004) . Also, the N-terminal structure of oligomers was similar to that of monomeric A ⁇ .
  • Fc ⁇ RIIb binds the N-terrainal region of A ⁇ , and identified first the binding site structures between Fc ⁇ RIIb and A ⁇ using the N- terminal structure of A ⁇ , which was determined through a nuclear magnetic resonance (NMR) study of a computational prediction method (Affinity ® program: InsightII, Accelrys Inc) .
  • NMR nuclear magnetic resonance
  • Affinity ® program: InsightII, Accelrys Inc The structures of the binding regions in Fc ⁇ RIIb and A ⁇ were determined using a known crystal structure of FcyRIIb (Sondermann, P. et al . , EMBO J. 18: 1095-1103, 1999) .
  • Fc ⁇ RIIb When a tryptophan residue critical for the binding of Fc ⁇ RIIb to IgG was replaced with alanine, Fc ⁇ RIIb showed remarkably reduced binding affinity to A ⁇ .
  • the structure prediction was carried out by placing the N- terminal region of A ⁇ proximate into a tryptophan pocket of Fc ⁇ RIIb.
  • a ⁇ i-42 Phe4 was first artificially located closed to Trp92 and Trpll5 residues of hFc ⁇ RIIb, and the general binding procedure was then performed as follows. Molecular dynamic calculations for the binding between hFc ⁇ RIIb and A ⁇ were carried out using the CVFF force field.
  • the initial structure was generated using a Monte Carlo minimization method, and simulated to generate actual non-bond contacts using a Cell Multipole method. Such simulated annealing started at 500 K, and the temperature was slowly cooled down to 300 K for stabilization through over 50 steps, followed by a final round of over 1000 steps of energy minimization for final structure calculation.
  • Example 10 Based on the results of Example 10, a sequence spanning from the first to ninth residues from the N- terminus of A ⁇ , and a 95 to 101 sequence and a 107 to 114 sequence of mouse Fc ⁇ RIIb, which are A ⁇ docking sites, were synthesized. Also, peptides, in which a residue involved in A ⁇ -c ⁇ RIIb binding in the above sequences was replaced with alanine, were synthesized. The peptides (wild type and mutant) have the sequences represented by SEQ ID Nos. 24 to 33 (FIG. 10a) . The synthesized peptides were individually allowed to react with a mixture of FcyRIIb- CD40 and A ⁇ (5 ⁇ M) or PBS. Then, luciferase activity was measured.
  • EXAMPLE 12 Inhibition of A ⁇ -induced neurotoxicity using the peptides inhibiting Fc ⁇ RIIb-A ⁇ binding
  • rat primary neuronal cells were treated with the peptides (15 ⁇ M each) prepared in Example 11 and A ⁇ (5 ⁇ M) for 48 hrs. Relative cell survival rates were then measured and compared with a control.
  • hippocampal neurons were treated with peptides #1 to #7
  • rat cortical neurons were treated with peptides #8 to #10.
  • binding inhibitory peptides were found to inhibit the neuronal toxicity induced by A ⁇ through its binding to
  • Fc ⁇ RIIb Fc ⁇ RIIb (FIG. 11) .
  • the peptides are able to strongly inhibit the neurotoxic signaling of A ⁇ , they may be useful in the prevention and treatment of AD.
  • mice were not used in this test because it takes a lot of time to breed the animals, and they are expensive. Instead, normal mice were used in this memory test because the same AD symptoms as in Tg2576 mice were observed when A ⁇ was injected into the brains of normal mice.
  • Normal BALB/c mice were injected intracerebro- ventricularly (i.c.v.) with A ⁇ (1.855 ⁇ g/5 ⁇ l, 410 pmole) alone or in combination with a specific peptide. After one day, a Y-maze test and a passive avoidance test were performed. Memory was assessed as described in Yan et al. ⁇ Br. J. Pharmacol. , 133:89-96, 2001) . When mice received i.c.v.
  • peptides #1 and #9 may be effective in the prevention and treatment of AD.
  • Brain specimens were prepared from the mice of Example 13 according to the same method as in Example 6. Sections were immunostained with primary antibodies, anti-A ⁇ antibody (Biosource, USA) plus an antibody to a marker of neurons, anti-neuron specific enolase (NSE) antibody
  • anti-mouse-FITC antibody goat anti-mouse IgG conjugated to FITC (Santa Cruz Biotechnology, USA)
  • anti- mouse-TRITC antibody goat anti-mouse IgG conjugated to TRITC (Santa Cruz Biotechnology, USA)
  • anti-rabbit- goat-anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA)
  • EXAMPLE 15-1 Analysis of change of apoptosis by knock-out of kinases through shRNAs
  • the present inventors analyzed degree of apoptosis after knocking out various kinases which are predicted to act downstream of Fc ⁇ RIIb using shRNAs against the kinase genes in order to investigate which molecules mediate intracellular signaling when oligomeric A ⁇ s bind to Fc ⁇ RIIb.
  • the inventors transfected mouse hippocampal cell line HT22 with pFc ⁇ RIIb prepared in the Example 3 and recombinant vectors prepared by inserting oligonucleotides encoding shRNA sequence specific for kinases Syk, Btk, Lyn and AbIl (SEQ ID NOs: 19 to 23, respectively, See Table 2) into pSM2c (Open Biosystems, USA) . After incubating for 24 hours, we observed inhibitory effects of shRNA of those kinases on apoptosis induced by Fc ⁇ RIIb.
  • EXAMPLE 15-2 Analysis of apoptosis change by kinase inhibitors
  • the present inventors measured degree of apoptosis using selective inhibitors of various intracellular kinases in order to confirm whether the activities of the kinases resulted from binding between oligomeric A ⁇ and Fc ⁇ RIIb.
  • the inventors treated a mouse hippocampal cell line, HT22, with LY294002 (Sigma, USA) which is an inhibitor of PI3K, SP600125 (Sigma, USA) which is an inhibitor of JNK, LiCl (Sigma, USA) which is an inhibitor of SK3b/IMPase, L-690,330 Tocris, USA) which is an inhibitor of IMPase, PD98059 (Sigma, USA) which is an inhibitor of MEK, and SB203580 (Sigma, USA) which is an inhibitor of p38, respectively, in a dose of 5 ⁇ M (5 mM only for LiCl) .
  • the inventors treated the HT22 cells with the above kinase inhibitors and a Syk inhibitor, piceatannol (Sigma, USA) and 2 hours after the treatment the inventors treated the cells with 5 uM of A ⁇ i-42, in order to confirm whether said kinase inhibitors inhibit apoptosis due to oligomeric A ⁇ .
  • the apoptosis of experimental groups pretreated with LY294002, SP600125, LiCl and L- 690,330 and piceatnannol, respectively decreased, whereas no effect was seen in the experimental group pre-treated with PD98059 and SB203580, respectively (FIG. 15b) .
  • an interaction inhibitor is provided for effectively inhibiting the binding of A ⁇ to Fc ⁇ RIIb in neuronal cells and an animal model of Alzheimer's disease, thereby reducing A ⁇ -induced neurotoxicity and cell death therein.
  • the present inhibitor is useful in the diagnosis, prevention and treatment of Alzheimer's disease.

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Abstract

L'invention concerne un agent destiné à diagnostiquer, prévenir et traiter la maladie d'Alzheimer, ainsi qu'un procédé de criblage associé. En particulier, l'invention concerne un inhibiteur ou un antagoniste de FcγRIIb, sélectionné dans le groupe comprenant : un FcγRIIb soluble, des variants et des protéines de domaine extracellulaire de celui-ci, des anticorps anti-FcγRIIb, des peptides spécifiques à FcγRIIb, des ARNsi spécifiques à FcγRIIb, et des inhibiteurs de l'activité ou de l'expression de kinases intracellulaires régulées par FcγRIIb; un tel inhibiteur ou antagoniste de FcγRIIb pouvant être utilisé pour diagnostiquer, prévenir et traiter la maladie d'Alzheimer par inhibition d'une transduction de signal, d'une internalisation cellulaire, d'une toxicité neuronale, d'une apoptose et d'une perte de mémoire, cette inhibition étant réalisée par inhibition de la liaison qui relie FcγRIIb à Aβ.
EP08848333.4A 2007-11-09 2008-11-07 Compositions et procede permettant de diagnostiquer, de prevenir et de traiter la maladie d'alzheimer Active EP2268296B1 (fr)

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PCT/KR2008/006570 WO2009061147A1 (fr) 2007-11-09 2008-11-07 Compositions et procédé permettant de diagnostiquer, de prévenir et de traiter la maladie d'alzheimer

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KR20110119513A (ko) 2010-04-26 2011-11-02 한국전자통신연구원 포토트랜지스터를 이용한 알츠하이머 질병의 조기 진단 방법
US9023607B2 (en) 2010-04-26 2015-05-05 Intellectual Discovery Co., Ltd. Method for early diagnosis of alzheimer'S disease using phototransistor
KR101352670B1 (ko) * 2010-06-15 2014-01-17 울산대학교 산학협력단 알츠하이머 전구단백질 분비 펩타이드의 축적물을 포함하는 뇌 노화 감별 진단 마커
US9541561B2 (en) 2012-06-14 2017-01-10 Electronics And Telecommunications Research Institute Method for diagnosing Alzheimer's disease using biomaterial
KR101987272B1 (ko) * 2017-09-15 2019-06-10 국민대학교 산학협력단 Fc 감마 수용체 변이체 MG2A28.1
KR101987292B1 (ko) * 2017-10-12 2019-06-10 국민대학교 산학협력단 Fc 감마 수용체 변이체 MG2B45.1
KR101987268B1 (ko) * 2017-10-12 2019-06-10 국민대학교 산학협력단 Fc 감마 수용체 변이체 MG2A45
KR101986252B1 (ko) * 2017-09-15 2019-06-05 국민대학교 산학협력단 Fc 감마 수용체 변이체 MG2A28
KR101987279B1 (ko) * 2017-10-12 2019-06-10 국민대학교 산학협력단 Fc 감마 수용체 변이체 MG2A45.1
KR20210048136A (ko) 2019-10-23 2021-05-03 주식회사 에이아이플랫폼 망막 영상 데이터 기반의 알츠하이머치매 분석 방법
KR20210048135A (ko) 2019-10-23 2021-05-03 주식회사 에이아이플랫폼 망막 영상 데이터 기반의 알츠하이머치매 분석 시스템
KR102351126B1 (ko) * 2019-12-03 2022-01-13 재단법인대구경북과학기술원 App 및 mdga1 단백질 상호작용 억제제를 포함하는 약학적 조성물 및 이를 이용한 스크리닝 방법

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EP2268296B1 (fr) 2015-08-12
EP2268296A4 (fr) 2012-04-25
US20120114669A1 (en) 2012-05-10
KR101397554B1 (ko) 2014-05-21
US20090123459A1 (en) 2009-05-14
US8124358B2 (en) 2012-02-28
WO2009061147A1 (fr) 2009-05-14
KR20090048192A (ko) 2009-05-13

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